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Epigenetics Reshapes the Therapeutic Arena

Structure-based design used to map the entirety of the BET binding pocket and design best-in-class BET inhibitors. [Constellation Pharmaceuticals]

A term originally coined by Conrad Hal Waddington in the 1940s to describe the causal relationship between genotypes and phenotypes, epigenetics has witnessed profound transformations as a concept.

The increasing appreciation of the roles that epigenetic changes play in a growing number of medical conditions opened the attractive possibility to therapeutically modulate them, and the approval of the first four epigenetic compounds set the stage for a new era that promises to reshape the therapeutic arena.

“We are pursuing drug discovery in a variety of areas relevant to chromatin structure and function, and this is the key feature that allowed us to establish our industry-leading programs in these areas,” says Jim Audia, Ph.D., CSO at Constellation Pharmaceuticals.

The most advanced of the epigenetic drug discovery efforts at Constellation, the BET (bromodomain and extra-terminal) program, focuses on small molecule inhibitors of the BET family proteins. In mammals, the BET family includes the ubiquitously expressed Brd2, Brd3, and Brd4, and the testis-specific isoform BRDT, which function to facilitate gene activation.

Each of these proteins has two N-terminal bromodomains, which harbor a deep, hydrophobic pocket that recognizes specific acetylated lysine residues on histone tails. “Their very well-formed pocket allows us to use structure-based drug design to find binding sites for small molecule inhibitors,” says Dr. Audia.

While pursuing this work, other researchers discovered that BET proteins selectively regulate the NF-κB pathway in inflammation, prompting Constellation to pursue this mechanism in oncology due to the importance of NF-κB-dependent transcriptional signaling in many malignant tumors.

One of the surprising findings was that BET proteins, particularly Brd2 and Brd4, localize to the transcriptional start sites of the myc oncoprotein, downregulating its expression in a variety of cellular contexts. The possibility to directly displace them by small molecule inhibitors promises opportunities to rapidly and potently suppress myc transcription in an oncology setting.

“We found a relatively broad subset of hematological malignancies that appear to be very sensitive to bromodomain inhibitors, and we are now optimizing our initial prototypic targets to ultimately make them become clinically relevant molecules,” explains Dr. Audia. This work, in addition, promises to elucidate in more detail the biology of the proteins involved in chromatin remodeling.

“We are learning how context-dependent everything is, how subtly different the effects of some of these targets may be depending on the cell type and on the state of the cell. One of our goals, that we think is both possible and advantageous, and perhaps even necessary, is to understand the factors that control this chromatin-based signaling,” reveals Dr. Audia.

Constellation recently reported its collaboration with the Leukemia & Lymphoma Foundation to support preclinical and Phase I programs that explore the therapeutic potential of BET bromodomain inhibitors in hematological malignancies.

“In addition, we have an exclusive collaboration with Genentech, a broad effort that is being driven by the belief that a number of targets across various methyltransferases, demethylases, and bromodomain readers offer the potential for significant therapeutic applications,” says Keith Dionne, Ph.D., president and CEO at Constellation.

The collaboration is not restricted to therapeutic targets in malignant tumors. This reflects the vast body of experimental findings that underscore the key roles that epigenetic modifications play in medical conditions outside the sphere of oncology such as inflammatory, neurodegenerative, and cardiovascular diseases.

“Rapid progress in these areas is driven by a partnership between chromatin biology and chemistry, which work together to understand the specificity of the interaction from the chemistry side, and the therapeutic impact from the biology side,” explains Dr. Dionne.

Inhibiting EZH2

“GlaxoSmithKline plans to take EZH2 inhibitors into the clinic in cancer,” says Peter J. Tummino, Ph.D., head of biology, cancer epigenetics discovery performance unit at GlaxoSmithKline (GSK). EZH2, the catalytic component of the polycomb repressive complex 2 (PRC2), represses gene transcription by methylating lysine 27 residues on the histone H3 tails. EZH2 overexpression has been reported in several types of solid tumors while, in contrast, mainly EZH2 point mutations were described in lymphomas.

“We found that these heterozygous mutations are biochemically neomorphic, an aspect that was not obvious when the mutations were discovered,” explains Dr. Tummino.

The presence of these gain-of-function EZH2 mutations pointed toward a desire to therapeutically inhibit the activity of the protein. Previously, scientists at GSK have developed several new reagents that helped conduct two EZH2 high-throughput screens. “We have been able to develop potent and selective small molecule EZH2 inhibitors that inhibit histone H3 lysine 27 trimethylation and possess potent antiproliferative activity specifically in cells with EZH2 activating mutations,” says Dr. Tummino.

Most recently, scientists at GSK described GSK126, a small molecule EZH2 inhibitor that can reactivate silenced PRC2 target genes. GSK126 inhibited the proliferation of diffuse large B-cell lymphoma cell lines harboring a heterozygous Y641 gain-of-function mutation, which is the most frequently encountered EZH2 mutation in this group of malignancies, and inhibited the growth of EZH2 mutant diffuse large B-cell lymphoma mouse xenografts.

Achieving Specificity

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According to RaNA Therapeutics, its oligos interrupt PRC2 recruitment.

“Our approach differs from the one used by many other epigenetic companies, in that we specifically target individual genes to upregulate,” says Arthur Krieg, M.D., president and CEO at RaNA Therapeutics. Investigators at RaNA achieve this specificity by targeting individual noncoding RNA molecules that bind PRC2 to repress the expression of individual genes.

“Oligonucleotide therapeutics are particularly well suited for this, because we can take advantage of the Watson-Crick base-pairing to specifically target individual genes, and with the knowledge coming out on noncoding RNAs, we can now accomplish this in a highly selective fashion,” notes Dr. Krieg.

One of the target genes that Dr. Krieg and colleagues used to illustrate this proof of concept is erythropoietin. “By using an oligonucleotide to target a noncoding RNA, we were able to prevent PRC2 recruitment to the erythropoietin locus, derepress gene expression, and upregulate erythropoietin expression in vitro and in vivo,” explains Dr. Krieg. Investigators at RaNA are planning to use this approach as they proceed toward clinical development with several therapeutic targets.

“We live in the next frontier in genetics and genomics, and one of the big advances has been the understanding that epigenetics is involved, in an ongoing basis, in regulating the expression of most genes in the genome in a highly selective and tissue-specific manner,” explains Dr. Krieg.

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